Receptor tyrosine kinase assays

ABSTRACT

Methods for detecting phosphorylation of receptor tyrosine kinases (“RTKs”) upon activation and the modulation of activation by a candidate compound are provided. The method employs cells comprising two fusion products: (1) an RTK fused to a small fragment of β-galactosidase and (2) a phosphotyrosine binding peptide fused to the large fragment of β-galactosidase, where the 2 fragments weakly complex to form an active enzyme, and optionally a construct for a cytosolic RTK phosphorylating kinase, when the RTK does not autophosphoryate. To detect phosphorylation a β-galactosidase substrate is added to the cells, whereby product formation indicates the occurrence of phosphorylation.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims priority from U.S. patent application Ser. No.12/536,667, filed Aug. 6, 2009 and from U.S. Provisional PatentApplication No. 61/089,799, filed Aug. 18, 2008, both of which arehereby incorporated by reference in their entirety.

STATEMENT OF GOVERNMENTAL SUPPORT

None.

REFERENCE TO SEQUENCE LISTING, COMPUTER PROGRAM, OR COMPACT DISK

Applicants submit herewith a Sequence Listing in computer readable formin accordance with EFS Web Legal Framework. Applicants incorporate thecontents of the sequence listing by reference in its entirety.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The field of this invention is the determination of phosphorylation oftyrosine on a receptor tyrosine kinase and screening of compounds thataffect the phosphorylation.

2. Background

Presented below is background information on certain aspects of thepresent invention as they may relate to technical features referred toin the detailed description, but not necessarily described in detail.These materials may be consulted for specific language, which may beomitted from the present specification and are, as stated in theConclusion, incorporated by reference. The discussion below should notbe construed as an admission as to the relevance of the information tothe appended claims or the prior art effect of the material described.

Pharmaceutical small molecule drug discovery is predicated ondiscovering compounds that bind to receptors or cytosolic proteins andact as agonists, antagonists, inverse agonists or modulators. Oneimportant class of proteins known as receptor tyrosine kinases (“RTKs”)are attractive targets, as these proteins act to induce a number ofdisease associated pathways. An important focus of pharmaceutical drugdiscovery is the identification of surrogate ligands for proteins, e.g.,receptors, kinases, or other proteins in the pathway of phosphorylation.Of particular interest in this respect is a subclass of cell surfacereceptor proteins known as receptor tyrosine kinases. Another importantclass of proteins is the cytosolic kinases, which can phosphorylate oneor a plurality of RTKs. By activating or inhibiting these kinases, onecan inhibit the activation of the RTK target of the cytosolic kinase.

The RTK family functions in the regulation of cell growth, celldifferentiation, adhesion, migration and apoptosis (Blume-Jensen andHunter 2001 Nature 411:355-65) (Ullrich and Schlessinger 1990 Cell61:203-12) (Schlessinger 2000 Cell 103:211-25) (Hubbard and Till 2000Annu Rev Biochem 69:373-98). A number of human diseases have been linkedto alterations in RTKs (Akin and Metcalfe 2004 J Allergy Clin Immunol114:13-9) (Verheul and Pinedo 2003 Drugs Today (Barc) 39 Suppl C: 81-93)(Corfas et al., 2004 Nat Neurosci 7:575-80). Many RTKs have beenidentified as oncogenes in transforming retrovirus or human cancers(Hunter 2000 Cell 100:113-27) (Shawver et al., 2002) (Muller-Tidow etal., 2004), and recent reports indicate that RTKs may play a criticalrole in almost all types of human cancer (Shawver et al., 2002 CancerCell 1:117-23) (Prenzel et al., 2000 Breast Cancer Res 2:184-90) (Mass2004 Int J Radiat Oncol Biol Phys 58:932-40). Both naturally occurringand artificial ligands that modulate RTK activity and signaling thuswould be of tremendous interest from a therapeutic standpoint withrespect to cancer and other diseases. (Haluska and Adjei 2001 Curr OpinInvestig Drugs 2:280-6) (Sawyer et al., 2003 BioTechniques Suppl:2-10,12-5). The ability to quickly, efficiently, and effectively screen vastlibraries of compounds for particular activities has become a goal ofthe pharmaceutical industry. Desirably, the methods provide more thanjust binding information, frequently employing whole cells, wherebiological processes occur in relation to the compounds being screened.

Many cytokine receptors do not possess intrinsic kinase activity.However, they also initiate intracellular cascades of tyrosinephosphorylation. To do this they interact with separate proteins thatare in the cytosol termed non-receptor tyrosine kinases (NRTK's). Theseproteins, such as the JAK kinases, bind to the intracellular domain ofcytokine receptors. Once the cytokine receptor binds ligand andoligomerizes, this brings the JAK proteins into close proximityinitiating trans-phosphorylation (by the JAK proteins) of the JAKproteins and the associated receptor.

High throughput screening has become a commonly employed strategy toidentify novel compounds with particular activities from a diversechemical library of compounds. Often, high throughput screening assaysare either based upon measuring compound binding to defined moleculartargets or measuring functional outputs resulting from compound/receptorinteractions. However, both binding assays and functional assays havelimitations. For example, for various technical reasons, binding assaysare preformed in non-physiological conditions. Artificial,non-physiological conditions may impact and influence receptorpharmacology, leading to increased unreliability and difficulty inaccurate interpretation of the data. Another drawback arises from thenature of the assay, which measures receptor binding only. Thus, bindingcompetition assays do not provide information regarding thephysiological function of ligands, such as whether the ligand functionsas an agonist or antagonist. Since the only information obtained isbinding, where the binding need not be at the target site, there can bemany false positives.

Functional assays overcome many of the limitations associated withbinding competition assays. Normally, cells are employed, which have thecapability to respond to agonist binding as part of the assay protocolTherefore, the assays can provide a measure of the activity resultingfrom binding and allow for activity/concentration determinations. Withthe assay being performed under physiological conditions within thecell, one obtains results that more closely approximate the results thatmay be anticipated in vivo.

Several functional assays have been described for receptor tyrosinekinases. Exemplary assays include the quantification ofautophosphorylation of RTKs (Olive 2004 Expert Rev Proteomics 1:327-41),measurement of phosphorylation of RTKs and downstream signalingmolecules (ibid), measurement of intracellular calcium release (Dupriezet al., 2002 Receptors Channels 8:319-30), or measurement of RTKdependent cell proliferation (Mosmann 1983 J Immunol Methods 65:55-63)(Bellamy 1992 Drugs 44;690-708). Despite the substantial variety ofassays that have been developed for evaluating ligands for RTKs, thereis still a substantial need for additional assays that can provideadvantages as to the nature of the protocol, the involvement of thetechnician in performing the assay, the number of steps that can lead toerrors in the result, the choice of equipment, the effect of organicsolvents, the dynamic range and the sensitivity of the assay.

Relevant Patent Literature

U.S. Patents and applications include U.S. Pat. Nos. 5,667,980;5,773,237; 5,976,893; a group of patents with the same disclosure U.S.Pat. Nos. 5,891,650, 5,914,237, 6,025,145, and 6,287,784; 6,413,730,2004/0038298, 2004/0161787; 2006/0199226; and 2008/0103107.

SUMMARY OF THE INVENTION

Mammalian cells are provided comprising at least two genetic expressionconstructs: a first construct of an RTK fused to a first member of apair of fragments of β-galactosidase; a second construct of apolypeptide, (“a phosphotyrosine binding peptide”) that binds to thephosphorylated RTK fused to the second fragment of β-galactosidase; andas appropriate, a third construct expressing a cytosolic kinasephosphorylating tyrosine receptor kinases. Upon stimulation of the RTK,the expression product of the second construct binds to thephosphorylated RTK bringing the two fragments into proximity to form anactive β-galactosidase, where phosphorylation may result from the RTK orthe cytosolic kinase. The two fragments have a low affinity for eachother, so that there is relatively low formation of β-galactosidase inthe absence of the binding of the two expression products. Addition of asubstrate for the β-galactosidase that produces a detectable productprovides a readout related to the degree of binding of the twoexpression products. Examples of these peptides and kinases are givenbelow.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a bar graph of the response in U2OS cells ofTropomyosin-Related Kinase A fused to a low affinity small fragment ofβ-galactosidase (TrkA-PK) and Src Homology 2 containing transformingprotein 1 fused to a complementary β-galactosidase fragment (SHC1-EA) tothe addition of Nerve Growth Factor (NGF); It shows that Activation ofTrkA-PK in U2OS cells causes recruitment of SHC1-EA phosphotyrosinebinding peptide resulting in increased enzyme activity. U2OS cellsexpressing the TrkA-PK and SHC1-EA fusion proteins were plated at10K/well in each well of a 384-well plate in serum-free medium with 0.1%FBS. The next day, the cells were treated with 100 ng/ml NGF or PBS+1%BSA for different time periods at room temperature and assayed usingPathHunter Detection reagent.

FIG. 2 is a graph of the dose response in U2OS cells of TrkA-PK andSHC1-EA to the addition of NGF. It shows that U2OS TRKA SHC1 cells showdose response to NGF. 5K/well U2OS TRKA SHC1 double-stable cells wereplated in each well of a 384-well plate in serum-free medium with 0.1%FBS. The next day, the cells were treated with different concentrationsof NGF for 1 hr at room temperature. Then PathHunter chemiluminescentsubstrate was added and the signal was read 1 hr later. EC50 of 9.3ng/ml and an assay window of 7.8 were obtained.

FIG. 3 is a graph of the dose response resulting from inhibitors addedto U2OS cells of TrkA-PK and SHC1-EA followed by the addition of NGF. Itshows that TRK inhibitors inhibit NGF stimulated assay signal. 5K/wellU2OS TRKA SHC1 double-stable cells were plated in each well of a384-well plate in serum-free medium with 0.1% FBS. The next day, thecells were treated with different concentrations of TrkA inhibitor orK-252a for 10 min at room temperature followed by 20 ng/ml NGFstimulation for 1 hr at room temperature. Then PathHunterchemiluminescent substrate was added and the signal was read 1 hr later.TrkA inhibitor gave an IC50 of 18 nM and an assay window of 6.7. K-252agave an IC50 of 37 nM and an assay window of 7.6.

FIG. 4 is a bar graph of the dose response in U2OS of Platelet-DerivedGrowth Factor Receptor Beta fused to a low affinity small fragment ofβ-galactosidase (PDGFRB-PK) with different SH2 (Src Homology 2)domain-EA conjugates after treatment with Platelet Derived Growth FactorAB (PDGF-AB). It shows that PDGFRB interacts with different SH2domain-containing cytoplasmic proteins (phosphotyrosine bindingpeptides). 5K/well PDGFRB-PK SH2-containing protein-EA double-stablecells were plated in each well of a 384-well. The next day, the cellswere treated with (solid bars) or without (open bars) 100 ng/ml PDGF-ABfor 1 hr at room temperature. Then PathHunter® chemiluminescentsubstrate was added and the signal was read 2 hrs later. (PathHunter® isa trademark of DiscoveRx Corporation, Fremont, Calif.)

FIG. 5 is a graph of the dose response in U2OS cells of IGF1R fused to alow affinity small fragment of β-galactosidase (IGF1R-PK). Cells wereplated in a 384-well plate at 10,000 cells/well, stimulated with IGF1(Peprotech, Inc., Rocky Hill, N.J., Cat #/AF-100-11), a known ligand forIFG1R for 3 hours at room temperature according to the assay procedureprovided. Following stimulation, detection reagents were added andsignal was detected after 1 hour using the PathHunter® Detection Kit(93-0001). An assay window of 4.4 fold was observed and the EC50 for theligand IGF1 was 17 ng/ml.

FIG. 6 is a graph of the dose response in U2OS cell of insulin receptor(INSR) fused to a low affinity small fragment of β-galactosidase(INSR-PK). Cells were plated in a 384-well plate at 10,000 cells/well,stimulated with insulin a known ligand for INSR, for 3 hours at roomtemperature according to the assay procedure provided. Followingstimulation, detection reagents were added and signal was detected after1 hour using the PathHunter® Detection Kit (93-0001). An assay window of7.6 fold was observed and the EC50 for the ligand insulin was 2.0 ng/ml.

FIG. 7 is a graph of the dose response in U2OS cells of TrkB fused to alow affinity small fragment of β-galactosidase (TrkB-PK). Cells wereplated in a 384-well plate at 10,000 cells/well stimulated with BDNF(Peprotech, Cat #/450-02), a known ligand for TrkB, for 3 hours at roomtemperature according to the assay procedure provided. Followingstimulation, detection reagents were added and signal was detected after1 hour using the PathHunter® Detection Kit (93-0001). An assay window of4.0 fold was observed and the EC50 for the ligand BDNF was 4.21 ng/ml.

FIG. 8 is a graph of the dose response in U2OS cells of TrkC fused to alow affinity small fragment of β-galactosidase (TrkC-PK). Cells wereplated in a 384-well plate at 10,000 cells/well, stimulated with NT3(Peprotech, Cat #/450-03), a known ligand for TrkC, for 3 hours at roomtemperature according to the assay procedure provided. Followingstimulation, detection reagents were added and signal was detected after1 hour using the PathHunter® Detection Kit (93-0001). An assay window of8.1 fold was observed and the EC50 for the ligand NT3 was 7 ng/ml.

FIG. 9 is a graph of the dose response in U2OS cells of G-CSFR fused toa low affinity small fragment of β-galactosidase (CSF3R-PK). The uppercurve is the response in the presence of over expression of Jak2 and thelower curve (squares) is the response in the absence of over expressionof Jak2. Cells were plated in a 384-well plate at 10,000 cells/well,stimulated with G-CSF (Peprotech 300-23), a known ligand for G-CSFR, inPBS+0.1% BSA, for 3 hours at room temperature according to the assayprocedure provided. Following stimulation, detection reagents were addedand signal was detected after 1 hour using the PathHunter® Detection Kit(93-0001). An assay window of 7 fold was observed and the EC₅₀ for theligand G-CSF was 4 ng/ml.

DESCRIPTION OF THE SPECIFIC EMBODIMENTS

Methods are provided for determining the phosphorylation of RTKs. Doubleor treble stable transformed cells are employed comprising two, andoptionally a third, expression constructs: (1) a fusion of an RTK with amember of an enzyme fragment pair at the cytosolic C-terminus of theRTK; (2) a polypeptide sequence that binds to the phosphorylated RTK (aphosphotyrosine binding domain) fused to the complementary member of theenzyme fragment pair; and where the RTK does not auto-phosphorylate, (3)a non-receptor tyrosine kinase, generally with a strong promoter forover expression. The enzyme pair is derived from β-galactosidase, wherethe fragments are relatively unable to independently complex to form anactive enzyme, namely having a weak affinity for each other, but able toform an active β-galactosidase when the proteins bind together to whichthe fragments are fused.

In performing the method, the cells are grown in an appropriate medium.The cells are seeded in basal media with Bovine Serum Albumin (BSA),using commonly employed conditions. For determining whether a candidatecompound is an agonist or inverse agonist, the putative agonist is addedand the cells incubated for a sufficient time for a reaction to occur.For determining whether a candidate compound is an antagonist, the cellsare first incubated with the putative antagonist for sufficient time forthe antagonist to bind, followed by the addition of an agonist andincubation for sufficient time for a reaction to occur. Fordetermination of whether a candidate compound is a modulator, cells arefirst incubated with a limiting concentration of either an agonist orantagonist, followed by incubation with the proposed modulator to assessa change in the response. A commercially available β-galactosidasesubstrate is then added that provides a detectable signal, the substrateoptionally combined with a lysing agent, and the signal detected as ameasure of the binding activity of the agonist or antagonist.

The media will be conventional for the particular cells used; F-12 forCHO cells, modified Eagle's media for U2OS cells, standard DMEM for HEKcells, etc. Conveniently, the assays are performed in microtiter wellplates, where the volumes may vary from about 4 to 100 μl, more usually10 to 25 μl. Generally, about 2 to 20×10³ cells per 10 μl are employedin the assay, more usually 3 to 10×10³ cells per 10 μl are employed inthe assay.

Temperatures will generally range from about 10 to 40° C. With theagonist assay, ambient temperatures are convenient, while with theantagonist assay, physiologic temperature (37° C.) is convenient. Theincubation times employed with the ligands are to provide for a robustresult, generally ranging from about 5 min to 2 h, more usually fromabout 10 min to 1 h, where the ligand has sufficient time to bind to theRTK, phosphorylation to occur and binding of the PTB-comprising-proteinto the phosphorylated RTK in sufficient number to occur. (By PTB isintended all phosphotyrosine binding domains including domains referredto as phosphotyrosine domains, SH2 domains, artificially engineereddomains, single chain, e.g., Fab, antibodies, and the like.) The precisetime employed for achieving at least substantial optimization can bedetermined empirically.

With a β-galactosidase substrate able to cross the cell membrane, thesubstrate is added as a dissolving solid or in a solution.Alternatively, the reagent solution may provide for permeabilizing orlysis of the cells and release of any complex formed in the cell to theassay medium. Any conventional lysis buffer may be employed that doesnot interfere with the β-galactosidase reaction with its substrate.Various ionic buffers, such as CHAPS, may be employed at 1-5%, generallynot more than 3%, in a convenient buffer, such as PBS or HEPES, wherenumerous other substitutes are known in the field. The reagent solutionwill generally be about 0.5-2 times the volume of the assay medium.After addition of the β-galactosidase substrate, the solution willusually be incubated for from 5-200 min, usually 10-150 min and thesignal read. The temperature will generally be at the temperature of theincubation medium or conveniently in the range of about 20-40° C.

The β-galactosidase substrate will provide a fluorescent or luminescentproduct. A fluorescent or chemiluminescent reader, respectively, is thenused to read the signal. Desirably a luminescent reagent and optionallya signal enhancer are employed. The luminescent reagent will be in largeexcess in relation to the maximum amount of β-galactosidase that islikely to be formed. Conveniently, a luminescent substrate is used,available as Galacton Star from ABI in conjunction with the Emerald IIenhancer. Any equivalent luminescent substrate composition may beemployed. The substrate will be present in about 1 to 10 weight percent,while the enhancer will be present in about 10 to 30 weight percent ofthe reagent solution. These amounts will vary depending upon theparticular substrate composition employed. The reagent solution may beprepared as a 5-20× concentrate or higher for sale or the solids may beprovided as powders and dissolved in water at the appropriateproportions.

Standards will usually be used, whereby the signal is related to theconcentration of a known stimulator performed under the same conditionsas the candidate compound. A graph can be prepared that shows the changein signal with the change in concentration of the standard compound. Theassay is sensitive to EC₅₀ s of not greater than nanomolar of candidatecompound, generally sensitive to less than about 1 μM, in most casessensitive to less than about 500 nM, frequently sensitive to less than100 nM and can in many cases detect EC₅₀s of less than 50 nM. The S/B(signal/background) ratios are generally are at least about 2 fold, moreusually at least about 3 fold, and can be greater than about 50 fold.

Instead of screening compounds for agonist or antagonist activity, e.g.,an active ligand, one can screen physiological or other samples forligand activity, namely as a diagnostic tool. A sample is used in placeof the candidate compound and the assay is performed in the same way.Physiological samples may include blood, plasma, saliva, CSF, tissue,lysed cells, etc. The sample may be subject to prior treatment, such asfiltration, centrifugation, citration, heating, precipitation, etc. Theamount of sample will depend upon the anticipated level of the agonistor antagonist. The subject method has the advantage over an immunoassayin measuring only components that actively bind the RTK rather thanepitopic sites of the components.

For convenience kits can be provided. In the subject assays, the EAfusion protein may be provided as a construct for expression of EA to beintroduced into the cell or cells may be provided that are appropriatelymodified to provide EA in the cell. Generally, the kits would include aninsert with instructions for performing the assay. The instructions maybe printed or electronic, e.g., a CD or floppy disk. The kits find usein marketing the product and encouraging the use of the assay forresearch and commercial settings.

Various, known cell lines may be employed for the assay. Cell lines thatfind use include U2OS, CHO, HeLa, HepG2, HEK, and the like.

The RTKs may be divided into self-phosphorylating receptors andreceptors that require an independent kinase, where a large number ofcytosolic kinases are known that have a relatively narrow repertoire ofRTKs that each phosphorylates when the RTK is activated. Therefore, thesubject assays allow for the investigation of activity of compounds thatare ligands for the RTKs or activators or inhibitors of cytosolickinases, where the RTKs that are phosphorylated by the cytosolic kinaseare known

There are a large number of RTKs that initiate a number of differentpathways and new RTKs are likely to be discovered. The RTKs have beendivided into a number of classes as follows: RTK class I (EGF receptorfamily); II (insulin receptor family); III (PDGR receptor family); IV(FGF receptor family); V (VEGF receptor family); VI (HGF receptorfamily); VII (Trk receptor family); VIII (AXL receptor family); IX (AXLreceptor family); X (LTK receptor family); XI (TIE receptor family); XII(ROR receptor family); XIII (DDR receptor family); XV (KLG receptorfamily); XVI (RYK receptor family); and XVII (MuSK receptor family).

Each of the RTKs binds to one or more polypeptides havingphosphotyrosine binding (“PTB”) domains. A large class of proteins havewhat is referred to as the SH2 (Src homology 2) domain. These proteinsinclude Ab1, GRB2, RasGAP, STAT proteins, ZAP70, SHP2, PI3K,Phospholipase C γ form, CRK, SOLS, Shc, and Src. Other proteins includeFRS2, FE65, X11/MINT, NUMB, EPS8, RGS12, DAB, ODIN, JIP-1, ARH andICAP1. (Further information on these proteins may be found by searchingfor symbols that contain these abbreviations, as given in Pubmed and/orat genenames.org/cgi-bin/hgnc_search.pl.) Complete sequence informationand annotations of the gene symbols used here may also be obtained bythose skilled in the art from OMIM or Swiss-Prot.

The PTB proteins need not be from the same species as the RTK, so longas they have a sufficient binding affinity to provide for a robustassay. The entire PTB protein need not be used, so long as the fragmentthat is employed comprises the PTB domain and has the desired level ofaffinity for the RTK phosphotyrosine site.

The RTKs that depend upon cytosolic receptors include T and B-cellreceptors, integrins, interferon receptors, interleukin receptors, GP130associated proteins, etc. Among the families of receptors that findapplication in the subject invention, the following are illustrative.Single chain: EPOR, GHR, CFSR, PRLR, MPL; IFN Family: IFNAR1, 2, IFNGR1,2; γC Family: IL2RA, B, G, IL4R, IL2RG (Type 1 receptor), IL4R-IL13RA1(Type II receptor), IL7R, IL2RG, IL9R, IL15RA, IL2RB, IL10RA, B,IL12RB1, 2, IL13RA1; IL3 Family: IL3RA, CSF2RA, B, IL5RA, GP130 Family:IL6R, IL6ST, IL11RA, LIFR, OSMR, IL6GT, CNTFR, IL6ST, and LIFR.

Where a wild-type cytosolic kinase (NRTK) is not endogenously availableand is required for phosphorylation, in addition to the RTKs indicatedimmediately above and the polypeptides having a PTB domain, there willalso be expression of an exogenously introduced wild-type NRTK with astrong promoter to provide over expression of the NRTK. Theoverexpression can be determined empirically, but will usually provide alevel of the NRTK in substantial excess, 2-fold or more, of the level ofthe NRTK present.

Typically individual plasmids are employed each with its own antibioticresistance gene, except where one of the components is multiunit, theunits may be on the same vector, e.g., plasmid. The vectors areintroduced into the cells sequentially or simultaneously and thetransformed cells selected by means of their antibiotic resistance. Inorder to facilitate the process bicistronic vectors may be used thatinclude internal ribosome entry sites, such that both receptor subunitscan be expressed from the same vector.

The detection system is dependent upon the use of β-galactosidase enzymefragment complementation. In this system a small fragment ofβ-galactosidase and a larger fragment of β-galactosidase are employed,where the two fragments have a low affinity for each other. The smallfragment of β-galactosidase (“ED”) may have the naturally occurringsequence or a mutated sequence. Of particular interest are smallfragments of from about 36 to 60, more usually not more than 50, aminoacids. Desirably, the ED has a low affinity for the large fragment ofβ-galactosidase (“EA), so that there is little complexation between thelarge and small fragments in the absence of binding of the RTK and PTBpeptides. For further description of the small fragments, see U.S. Pat.No. 7,135,325. For further description of mutated EDs, see U.S. patentapplication publication no. 2007/0275397, both of which references areincorporated herein in their entirety as if set forth herein. The smallEDs and mutated EDs will generally have less than about 0.5, but atleast about 0.1, of the activity of the wild-type sequence in the assayof interest or an analogous assay, while having less than about 60% ofthe conventionally used commercial sequence of about 90 amino acids inthe absence of being fused to other proteins. For increasing affinitybetween the ED and EA, the longer EDs will be used and free of mutationsfrom the wild-type sequence. One can determine empirically for aspecific assay the desirable level of affinity of the two fragments,having a higher affinity when the affinity for the PTB peptide for theRTK is low.

Two expression constructs, and optionally a third, are employed: afusion of one β-galactosidase fragment with the RTK, usually the smallfragment; a fusion of the other β-galactosidase fragment with the PTBpeptide, usually the large fragment; and optionally, an expressionconstruct for an appropriate cytosolic kinase, particularly with astrong promoter. Conveniently, each protein is expressed from adifferent plasmid, with each plasmid having its own antibioticresistance gene. Where the receptor is composed of multiple subunits,each encoded by a separate gene, conveniently, one may express more thanone protein per plasmid using multiple promoters or bicistronic vectorsor IRES.

For expression constructs and descriptions of other conventionalmanipulative processes, see, e.g., Sambrook, Fritsch & Maniatis,“Molecular Cloning: A Laboratory Manual,” Second Edition (1989) ColdSpring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (herein“Sambrook et al., 1989”); “DNA Cloning: A Practical Approach,” Volumes Iand II (D. N. Glover ed. 1985); “Oligonucleotide Synthesis” (M. J. Gaited. 1984); “Nucleic Acid Hybridization” [B. D. Hames & S. J. Higginseds. (1985)]; “Transcription And Translation” [B. D. Hames & S. J.Higgins, eds. (1984)]; “Animal Cell Culture” [R. I. Freshney, ed.(1986)]; “Immobilized Cells And Enzymes” [IRL Press, (1986)]; B. Perbal,“A Practical Guide To Molecular Cloning” (1984).

The gene encoding the fusion protein will be part of an expressionconstruct. The gene is positioned to be under transcriptional andtranslational regulatory regions functional in the cellular host. Theregulatory region may include an enhancer, which may provide suchadvantages as limiting the type of cell in which the fusion protein isexpressed, requiring specific conditions for expression, naturally beingexpressed with the protein, and the like. In many instances, theregulatory regions may be the native regulatory regions of the geneencoding the protein, where the fusion protein may replace the nativegene, may be in addition to the native protein, either integrated in thehost cell genome or non-integrated, e.g., on an extrachromosomalelement.

As indicated, the β-galactosidase fragment joined to the RTK will befused at the C-terminus of the RTK, generally linked through a linkerthat conveniently has from 1 to 2 GGGS units. The large fragment fusedto the PTB peptide may be fused directly to the peptide terminus, eitherN- or C-terminus, or have a linker, the same or different from the smallfragment linker.

The following examples are offered by way of illustration and not by wayof limitation.

EXPERIMENTAL

All cells lines used were from DiscoveRx Corporation and express variousRTKs tagged with PK (42aa, DSLAVVLQRRDWENPGVTQLNRLAARPPFASWRNSEEARTDR)(SEQ ID NO: 2) in cells stably expressing SH2-EA (Large β-galactosidasefragment, Full length β-galactosidase deleted in amino acids 31-41).

EXAMPLE

-   Amino acids 1-583=SHC1-   Amino Acids 584-597=Linker-   Amino Acids 598-1589=Large β-galactosidase fragment (EA)

(SEQ ID NO: 1)MDLLPPKPKYNPLRNESLSSLEEGASGSTPPEELPSPSASSLGPILPPLPGDDSPTTLCSFFPRMSNLRLANPAGGRPGSKGEPGRAADDGEGIVGAAMPDSGPLPLLQDMNKLSGGGGRRTRVEGGQLGGEEWTRHGSFVNKPTRGWLHPNDKVMGPGVSYLVRYMGCVEVLQSMRALDFNTRTQVTREAISLVCEAVPGAKGATRRRKPCSRPLSSILGRSNLKFAGMPITLTVSTSSLNLMAADCKQIIANHHMQSISFASGGDPDTAEYVAYVAKDPVNQRACHILECPEGLAQDVISTIGQAFELRFKQYLRNPPKLVTPHDRMAGFDGSAWDEEEEEPPDHQYYNDFPGKEPPLGGVVDMRLREGAAPGAARPTAPNAQTPSHLGATLPVGQPVGGDPEVRKQMPPPPPCPGRELFDDPSYVNVQNLDKARQAVGGAGPPNPAINGSAPRDLFDMKPFEDALRVPPPPQSVSMAEQLRGEPWFHGKLSRREAEALLQLNGDFLVRESTTTPGQYVLTGLQSGQPKHLLLVDPEGVVRTKDHRFESVSHLISYHMDNHLPIISAGSELCLQQPVERKL GGGGSGGGGSLESMGVITDSLAVVARTDRPSQQLRSLNGEWRFAWFPAPEAVPESWLECDLPEADTVVVPSNWQMHGYDAPIYTNVTYPITVNPPFVPTENPTGCYSLTFNVDESWLQEGQTRIIFDGVNSAFHLWCNGRWVGYGQDSRLPSEFDLSAFLRAGENRLAVMVLRWSDGSYLEDQDMWRMSGIFRDVSLLHKPTTQISDFHVATRFNDDFSRAVLEAEVQMCGELRDYLRVTVSLWQGETQVASGTAPFGGEIIDERGGYADRVTLRLNVENPKLWSAEIPNLYRAVVELHTADGTLIEAEACDVGFREVRIENGLLLLNGKPLLIRGVNRHEHHPLHGQVMDEQTMVQDILLMKQNNFNAVRCSHYPNHPLWYTLCDRYGLYVVDEANIETHGMVPMNRLTDDPRWLPAMSERVTRMVQRDRNHPSVIIWSLGNESGHGANHDALYRWIKSVDPSRPVQYEGGGADTTATDIICPMYARVDEDQPFPAVPKWSIKKWLSLPGETRPLILCEYAHAMGNSLGGFAKYWQAFRQYPRLQGGFVWDWVDQSLIKYDENGNPWSAYGGDFGDTPNDRQFCMNGLVFADRTPHPALTEAKHQQQFFQFRLSGQTIEVTSEYLFRHSDNELLHWMVALDGKPLASGEVPLDVAPQGKQLIELPELPQPESAGQLWLTVRVVQPNATAWSEAGHISAWQQWRLAENLSVTLPAASHAIPHLTTSEMDFCIELGNKRWQFNRQSGFLSQMWIGDKKQLLTPLRDQFTRAPLDNDIGVSEATRIDPNAWVERWKAAGHYQAEAALLQCTADTLADAVLITTAHAWQHQGKTLFISRKTYRIDGSGQMAITVDVEVASDTPHPARIGLNCQLAQVAERVNWLGLGPQENYPDRLTAACFDRWDLPLSDMYTPYVFPSENGLRCGTRELNYGPHQWRGDFQFNISRYSQQQLMETSHRHLLHAEEGTWLNIDGFHMGIGGDDSWSPSVSAEFQLSAGRYHYQLVWCQK 

RTK Fusions include: ErbB-1 (EGFR), ErbB-2, ErbB3, ErbB4, INSR, IGF1R,IRR, PDGFRA, PDGFRB, CSF-1R, C-Kit, FGFR1, FGFR2, FGFR3, FGFR4, Flt3,VEGFR1 (Flt-1), VEGFR-2 (Flk-1/KDR), VEGFR-3 (Flt-4), C-Met, RON, TrkA,TrkB, TrkC, AXL, MER, SKY (TYRO3) (Dtk), LTK (TYK1), ALK, Tie-1, Tie-2,(TEK), DDR1, DDR2, MuSK, RET, EPHA1, EPHA2, EPHA3, EPHA4, EPHA5, EPHA6,EPHA7, EPHA8, EPHA9, EPHB1, EPHB2, EPHB3, EPHB4, EPHB5, EPHB6, CCK4(PTK7), ROS, AATYK1, AATYK2, AATYK3, ROR1, and ROR2.

Cell lines include: U2OS containing TrkA-PK fusion and SHC1-EA, U2OScontaining INSR (insulin receptor)-PK fusion and PLCG2 (Phospholipase C,gamma 2 (phosphatidylinositol-specific))-EA, U2OS containing IGF1R-PK(insulin like growth factor-1 receptor) and SHC1-EA, U2OS containingTrkB-PK fusion and SHC1-EA, TrkC-PK fusion and SHC1-EA, U2OS containingPDGFRB-PK fusion containing PLCG1 (Phospholipase C, gamma 1)-EA, U2OScontaining PDGFRB-PK fusion containing Grb2 (Growth factorreceptor-bound protein 2)-EA, U2OS containing PDGFRB-PK fusioncontaining PLCG2-EA, U2OS containing PDGFRB-PK fusion containing PTPN11(protein tyrosine phosphatase, non-receptor type11)-EA, U2OS containingPDGFRB-PK fusion containing SYK (spleen tyrosine kinase)-EA, ErbB4(v-erb-a erythroblastic leukemia viral oncogene homolog 4)-PK fusion andGrb2 (Growth factor receptor-bound protein 2)-EA.

Generally, the expression constructs for the fusion proteins includes atleast (in order of 5′ to 3′) a promoter, followed by the receptor or SH2domain, followed by a linker, followed by either the EA or PK. For allassays, 10,000 cells per well were seeded in 20 μL media and incubatedovernight in 0.1% BSA and appropriate basal media (F-12 or DMEM). Foragonist assays, 5 μL compound was added to cells and incubated at roomtemperature. For antagonist assays, 5 μL 5× compound was added to cellsand incubated at 37° C./5% CO₂ for 10 minutes, after which 5 μL 6×agonist was added and incubated for 60 minutes at room temperature.SH2-EA complex formation with the RTK was detected with 50% (v/v) ofPathHunter® Detection Reagent (Dx 93-0001, PathHunter reagents areavailable from DiscoveRx, Corp., Fremont, Calif.) (Lysis buffer activeingredient 1% CHAPS, Emerald II™ and Galacton Star™ are from AppliedBiosystems). Data was read on Packard Victor 2® or PerkinElmer ViewLux®readers or comparable instrumentation and analyzed using GraphPad Prism4® analysis software.

U2OS cells expressing the TrkA-PK and SHC1-EA fusion proteins wereplated at 10K cells/well in each well of a 384-well plate in serum-freemedium with 0.1% FBS. The next day, the cells were treated with 100ng/ml NGF in PBS+1% BSA or PBS+1% BSA for different time periods at roomtemperature and assayed using PathHunter (DiscoveRx) Detection reagent.The results are shown in FIG. 1.

5K cells/well U2OS TrkA-PK SHC1-EA double-stable cells were plated ineach well of a 384-well plate in serum-free medium with 0.1% FBS. Thenext day, the cells were treated with different concentrations of NGFfor 1 hr at room temperature (see above). Then PathHunterchemiluminescent substrate was added and the signal was read 1 hr later.EC₅₀ of 9.3 ng/ml and an assay window of 7.8 were obtained. The resultsare reported in FIG. 2.

5K cells/well U2OS TrkA-PK SHC1-EA double-stable cells were plated ineach well of a 384-well plate in serum-free medium with 0.1% FBS. Thenext day, the cells were treated with different concentrations ofantagonists, such as a commercially available TrkA inhibitor or the Trkinhibitor K252a[8R*,9S*,11S*)-(−)-9-hydroxy-9-methoxycarbonyl-8-methyl-2,3,9,10-tetrahydro-8,11-epoxy-1H,8H,11H-2,7b,11a-triazadibenzo(a,g)cycloocta(cde)-trinden-1-one]for 10 min at room temperature followed by 20 ng/ml NGF stimulation for1 hr at room temperature (see above). Then PathHunter chemiluminescentsubstrate was added and the signal was read 1 hr later. TrkA inhibitorgave an IC₅₀ of 18 nM and an assay window of 6.7. K-252a gave an IC₅₀ of37 nM and an assay window of 7.6. The results are reported in FIG. 3.

5K cells/well PDGFRB-PK SH2-containing protein-EA double-stable cellswere plated in each well of a 384-well plate in serum-free medium with0.1% FBS. Six SH2-containing protein-EAs were used: SHC1-EA, Grb2-EA,PLCG-1-EA, PLCG2-EA, PTPN11-EA, and SYK-EA. The next day, the cells weretreated with or without 100 ng/ml PDGF-AB in serum-free medium with 0.1%FBS for 1 hr at room temperature. Then PathHunter chemiluminescentsubstrate was added and the signal was read 2 hrs later. The results arereported in FIG. 4.

10K cells/well IGF1R-PK SH2-containing protein-EA double stable cellswere plated in a 384-well plate, stimulated with IGF1 (Peprotech, Cat#/AF-100-11), a known ligand for IFG1R for 3 hours at room temperatureaccording to the assay procedure provided. Following stimulation,detection reagents were added and signal was detected after 1 hour usingthe PathHunter® Detection Kit (93-0001). An assay window of 4.4 fold wasobserved and the EC₅₀ for the ligand IGF1 was 17 ng/ml. The results arereported in FIG. 5.

10 k cells/well INSR-PK SH2-containing protein-EA double stable cellswere plated in a 384-well plate, stimulated with insulin, a known ligandfor INSR, for 3 hours at room temperature according to the assayprocedure provided. Following stimulation, detection reagents were addedand signal was detected after 1 hour using the PathHunter® Detection Kit(93-0001). An assay window of 7.6 fold was observed and the EC₅₀ for theligand IGF1 was 2.0 ng/ml.

10K cells/well TrkB-PK SH2-containing protein-EA double stable cell wereplated in a 384-well plate, stimulated with BDNF (Peprotech, Cat#/450-02), a known ligand for TrkB for 3 hours at room temperatureaccording to the assay procedure provided. Following stimulation,detection reagents were added and signal was detected after 1 hour usingthe PathHunter® Detection Kit (93-0001). An assay window of 4.0 fold wasobserved and the EC₅₀ for the ligand BDNF was 4.21 ng/ml. The resultsare reported in FIG. 7.

10K cells/well TrkC-PK SH2-containing protein-EA double stable cellswere plated in a 384-well plate, stimulated with NT3 (Peprotech, Cat#/450-03), a known ligand for TrkC for 3 hours at room temperatureaccording to the assay procedure provided. Following stimulation,detection reagents were added and signal was detected after 1 hour usingthe PathHunter® Detection Kit (93-0001). An assay window of 8.1 fold wasobserved and the EC₅₀ for the ligand NT3 was 7 ng/ml. The results arereported in FIG. 8.

There are a significant number of TKRs that depend upon cytosolictyrosine kinases for phosphorylation. The assays for activation of suchTKR receptors are substantially the same as described above, except thatthe cells are triply stable having an expression construct overexpressing the cytosolic tyrosine kinase. In the following example, U2OScells containing the following constructs were used: G-CSFR(granulocyte-colony stimulating factor receptor)-PK with neo selection;SHC1-EA with hygro selection and Jak2 with puromycin selection. Theresults are reported in FIG. 9.

The genes for the RTK, SH2 and NRTK domains may be obtained from anyconvenient source; commercial supplier, RT PCR from RNA isolated inaccordance with conventional procedures using known sequences as probes,and PCR from genomic DNA using primers from known sequences. The genesare PCR amplified to remove the stop codon at the 3′ end and thendigested with restriction enzymes where the restriction site is includedwith the primer sequences. These products are then purified inconventional ways and then ligated into a commercial vector into whichthe PK or EA has been inserted, in reading frame with the PK or EA.Separating the PK and the EA from the gene is a gly-ser linker thatprovides flexibility to the fusion proteins to enhance complementation.This linker is not required for activity. The transcriptional regulatoryregion is generally present in commercial vectors, such as the 5′LTR ofthe virus used for the vector. Alternatively, the CMV promoter may beused. The resulting vector is then introduced into the host cell byliposome mediated transfection or retrieval infection with Moloneymurine leukemia virus vector and packaging cell lines. The resultingvirus is then used for viral infection. The vectors also includeselection genes, such as hygromycin, puromycin and neomycin resistanceand cells into which the construct is integrated are selected in aconventional selection medium. The surviving cells are then screened inan agonist dose response assay using the Path-Hunter® Detection Kitreagents in white-walled microplates.

It is evident from the above results that the subject method providesfor a robust accurate assay for measuring agonists and antagonists forRTKs. Cells are provided that can be used effectively in high throughputscreening in a cellular environment, so as to closely define the effectof candidate compounds in a mammalian environment. The protocols areeasy, use standard equipment and can be readily automated.

CONCLUSION

Although the invention has been described with reference to the aboveexamples, it will be understood that modifications and variations areencompassed within the spirit and scope of the invention. Accordingly,the invention is limited only by the following claims. All referencesreferred to in the specification are incorporated by reference as iffully set forth therein.

What is claimed is:
 1. A method for determining an effect of a candidate compound on phosphorylation of a receptor tyrosine kinase (“RTK”) said method comprising: (a) providing a cell comprising (i) a first expression construct expressing a fusion of an RTK fused to a first enzyme fragment and (ii) a second expression construct expressing a fusion of a phosphotyrosine binding peptide fused to a second enzyme fragment, (b) wherein said first enzyme fragment and said second enzyme fragment are fragments of β-galactosidase that have low affinity for each other but when brought together by the binding of said RTK to said phosphotyrosine binding peptide form an active β-galactosidase, with the proviso that when said RTK does not autophosphorylate, in the absence of an endogenous active cytosolic tyrosine kinase, a third expression construct is included expressing a cytosolic tyrosine kinase to phosphorylate said RTK; (c) contacting said cell with said candidate compound; (d) incubating said cell for sufficient time for any phosphorylation to occur; (e) adding a β-galactosidase substrate to said cell, wherein said substrate forms a detectable product; and (f) detecting a product formed as indicative of the effect of the candidate compound on the phosphorylation of said RTK.
 2. A method according to claim 1, wherein said first enzyme fragment is the small fragment of β-galactosidase having fewer than 50 amino acids.
 3. A method according to claim 2, wherein said small fragment is mutated with respect to native β-galactosidase.
 4. A method according to claim 1, wherein said cell is a mammalian cell.
 5. A method according to claim 1, including the additional step of lysing said cell before said detecting.
 6. A method according to claim 1, wherein said product is chemiluminescent.
 7. A method according to claim 1, wherein said candidate compound is tested as an antagonist, wherein a ligand for said RTK is added prior to addition of said candidate compound.
 8. A method for determining presence of active ligand for receptor tyrosine kinase (“RTK”) in a sample, comprising: (a) providing a cell comprising (i) a first expression construct expressing a fusion of an RTK fused at its C-terminus to a first enzyme fragment and (ii) a second expression construct expressing a fusion of a phosphotyrosine binding peptide fused to a second enzyme fragment; (b) wherein said first enzyme fragment and said second enzyme fragment are fragments of β-galactosidase that have low affinity for each other but when brought together by the binding of said RTK to said phosphotyrosine binding peptide form an active β-galactosidase, with the proviso that when said RTK does not autophosphorylate, in the absence of an endogenous active cytosolic tyrosine kinase, a third expression construct is included expressing a cytosolic tyrosine kinase to phosphorylate said RTK; (c) contacting said cell with said sample; (d) incubating said cell for sufficient time for any phosphorylation to occur; (e) adding a β-galactosidase substrate to said cell, wherein said substrate forms a detectable product; and (f) detecting a product formed as indicative of the presence of an active ligand for said RTK.
 9. A method according to claim 8, wherein said first enzyme fragment is the small fragment of β-galactosidase having fewer than 50 amino acids.
 10. A method according to claim 9, wherein said small fragment is mutated with respect to native β-galactosidase.
 11. A method according to claim 8, wherein said cell is a mammalian cell.
 12. A method according to claim 8, including the additional step of lysing said cell before said detecting.
 13. A method according to claim 8, wherein said product is chemiluminescent. 